title: Importance of recognition loops B and D in the activation of human 5-HT₃ receptors by 5-HT and meta-chlorophenylbiguanide.
abstract: The 5-HT₃ receptor is a cation selective member of the pentameric Cys-loop ligand-gated ion channels. While five subunits are known to exist, only two receptor subtypes have been significantly characterized: the homomeric receptor consisting of five A subunits and the heteromeric receptor containing both A and B subunits. The agonist recognition and activation of these receptors is orchestrated by six recognition loops three, A-C, on the principal subunit, and three, D-F, on the complementary subunit. In this study we have focused on the B loop of the principal subunit and loop D of the complementary subunit where aligned amino acids differ between the two subunits. A mutational analysis has been carried out using both 5-HT and m-chlorophenylbiguanide (mCPBG) to characterize receptor activation in the mutant receptors using two-electrode voltage clamp in Xenopus oocytes. The results show that the B loop W178I mutation of the 5-HT3A subunit markedly reduces the efficacy of mCPBG in both the homomeric and heteromeric receptors, while activation by 5-HT remains intact. Replacement of the D loop amino acid triplet RQY of the 5-HT3A subunit, with the aligned residues from the 5-HT3B subunit, QEV, converts 5-HT to a weak partial agonist in both the homomer and heteromer, but does not compromise activation by mCPBG. Exchange of the RQY triplet for the 5-HT3B subunit homologue, QEV, increases the Hill coefficient and decreases the EC₅₀ of this mutant when expressed with the wild type 5-HT3A subunit.
authors.affiliation: Department of Pharmacology, University of Alberta, 9-70 Medical Sciences Building, Edmonton, AB, Canada T6H 2H7.
authors.forename: S D
authors.initials: SD
authors.lastname: Michaelson
authors.forename: I M
authors.initials: IM
authors.lastname: Paulsen
authors.forename: J L
authors.initials: JL
authors.lastname: Kozuska
authors.forename: I L
authors.initials: IL
authors.lastname: Martin
authors.forename: S M J
authors.initials: SM
authors.lastname: Dunn
mesh_terms: D000818:Animals; D001645:Biguanides / Q000494:pharmacology*; D004252:DNA Mutational Analysis; D006801:Humans; D008564:Membrane Potentials / Q000187:drug effects / Q000235:genetics; D009865:Oocytes / Q000187:drug effects / Q000502:physiology; D021122:Protein Subunits / Q000187:drug effects / Q000502:physiology*; D044406:Receptors, Serotonin, 5-HT3 / Q000737:chemistry* / Q000235:genetics / Q000378:metabolism*; D012701:Serotonin / Q000494:pharmacology*; D058827:Serotonin 5-HT3 Receptor Agonists / Q000494:pharmacology*; D014981:Xenopus
chemical_list: D001645:Biguanides; D021122:Protein Subunits; D044406:Receptors, Serotonin, 5-HT3; D058827:Serotonin 5-HT3 Receptor Agonists; D012701:Serotonin; C065734:1-(3-chlorophenyl)biguanide
keywords: 5-HT; 5-HT(3)A receptor; 5-HT(3)AB receptor; 5-HT(3)ABR; 5-HT(3)AR; 5-hydroxytryptamine; 5-hydroxytryptamine type 3A receptor; 5-hydroxytryptamine type 3AB receptor; Receptor activation; mCPBG; meta-chlorophenylbiguanide; nAChR; nicotinic acetylcholine receptor; wild type; wt